12-oxo-15-oxy-a-norprogesterones



United States Patent r 3,332,985 12-0XO-IS-OXY-A-NORPROGESTERONESPatrick A. Diassi, Westfield, N.J., assignor, by mesne assignments, toE. R. Squibb & Sons, Inc;, New York, N.Y., a corporation of Delaware NoDrawing. Filed May 12, 1965, Ser. No. 455,339 4 Claims. (Cl. 260-488)This invention relates to and has for its object the provision of newphysiologically active compounds, and more particularly, compounds ofthe formula MUS ll:

acids), the cycloalkane carboxylic and the cycloalkene carboxylic acids.

The novel compounds of this invention are pharmacologically activesubstances which possess anti-androgenic activity (i.e., they inhibitthe actions of androgens), and which may be used in the treatment ofsuch conditions as hyperandrogenic acne.

The compounds may be formulated for such adminstration, theconcentration and/ or dosage being based on the activity of theparticular compound and the requirements of the patient.

To prepare the compounds of this invention, A-norprogesterone is firstsubjected to the action of enzymes of a microorganism of the genusColletotrichum under oxidizing conditions. This oxidation-can best beeffected either by including A-norprogesterone in an aerobic culture .ofthe microorganism, or by bringing together in an aqueous medium thecompounds, air, and enzymes of non-proliferating cells of themicroorganism. In general, the conditions of culturing theColletotrichum microorganism for the purposes of this invention are(except for the inclusion of A-norprogesterone to be converted), thesame as those of culturing various other microorganisms for theproduction of antibiotics, Vitamin B-12, and other like substances. Themicroorganism is grown aerobically in contact with (in or on) suitablefermentation medium. A suitable medium essentially comprises a source ofcarbon and energy. The latter may be a carbohydrate, for example,molasses, glucose, maltose, starch or dextrin, a fatty acid, a fatand/or the compound itself; Preferably, however, the medium includes anassimilable source of carbon and energy in addition to the steriod.Among the fats utilizable for the purpose of this invention are lardoil, soybean oil, linseed oil, cottonseed oil, peanut oil, coconut oil,corn oil, castor oil, sesame oil, crude palm oil, fancy mutton tallow,sperm oil, olive oil, tristearin, tripalmitin, triolein and trilaurin.

Among the fatty acids utilizable for the purpose of this invention arestearic acid, palmitic acid, oleic acid, linoleic acid and myristicacid.

examples 1 9 3,332,985 Patented July 25, 1967 The source of nitrogenousfactors utilizable for the purposes of this invention may be organic(e.g., soybean meal, cornsteep liquor, yeast extract, meat extract and/or distillers solubles) or synthetic (i.e., composed of simple,synthesizable organic or inorganic compounds, such as ammonium salts,alkali nitrates, amino acids, or urea).

An adequate sterile air supply should be maintained during fermentation,for example, by the conventional methods of exposing a large surface ofthe medium to air or by utilizing submerged aerated culture. Thecompound may be added to the culture during the incubation period, orincluded in the medium prior to sterilization or inoculation. Thepreferred (but not limiting) range of the concentration of the compoundinthe culture is about 0.01% to about 0.1%. The culture period (orrather the time of subjecting the compound to the action of the enzyme)may vary considerably, the range of about twenty-four to ninety-sixhours being feasible, but not limiting.

The microbial process described hereinabove yields, inter alia, 123,15a-dihydroxy-A-norprogesterone. The 12, IS-dihydroxy derivative isthen acylated as by treatment with an acylating agent, for example, anacid anhydride (i.e., acetic anhydride) or an acyl halide (e.g., acetylchloride) in a basic medium (i.e., in pyridine) to yield the12-hydroxy-l5-acyloxy 12,15-diacyloxy derivative of the instantinvention.

Alternatively, the IZ-hydroxy-lS-acyloxy derivatives ofA-norprogesterones, which are new compounds of this invention, may beoxidized as by treatment with an oxidizing agent, for example, chromicacid to yield the '12-keto-15-acyloxy derivatives of A-norprogesterone'keto-15-hydroxy-A-norprogesterone new final products of the instantinvention. The invention may be illustrated by the following EXAMPLE 12B,15u-dihydr0xy-A-norprogesterone A 150 gallon fermentor containing 1112 g. of, A- progesterone in media containing corn steep liquor, NH HPO CaCO yeast extract, dextrose and soy bean oil is inoculated withaculture of Colletotrichum linicolae NCTC (National Collection of TypeCultures) No. 1194 (obtainable from the Commonwealth MycologicalInstitute, Kew, Surrey, England). =After thirty-six hours, the wholebroth is filtered and the filtrate extracted with onehalf volume ofchloroform in a Podbielniak extractor. The chloroform solution is washedwith water, dried over sodinm sulfate and evaporated to a crystallinemass. Trituration with 3 l000 ml. of hexane removes color and oil. Thecrude dry weight is 101.6 g. This material is recrystallized fromacetone/hexane to give 67 g. of product which containslla,15a-dihydroxy-A-norprogesterone and12,9,l5a-dihydroxy-A-norprogesterone.

The separation of these components is accomplished by columnchromatography on 2300 g. alumina. Elution of the column with chloroformand continued elution with chloroform/methanol 200:1 and :1 gives 10.3g. of 12B,15a-dihydroxy-A-norprogesterone having the followingproperties: M.P. 244246 C. [ali) +40 20.30 mg./2 ml. CHCl A222? 16,400k115i? 2.89, 5.89, 6.00, 6.21, 7.92, 8.51, 9.39 and 11.73

Analysis.-Calcd for C H O (332.42): C, 72.26%; H, 8.49%. Found: C,72.29%; H, 8.58%.

234 my; 6 max.

3 EXAMPLE 2 1 218,1 Sa-dihyarOxy-A -norprogesterone 1 -ace tate Asolution of 103 mg. of 12B,15a-dihydroxyprogesterone in 5 ml. of drypyridine and 2 ml. of acetic anhydride is kept at room temperature forfour hours, then diluted with ice water and extracted with chloroform.The chloroform is washed with water, 2 N HCl, and water, and evaporatedto dryness. The residue is plate chromatographed on alumina (activity V)using chloroform as the developing solvent. Two bands are detectable byU'.V. at R;=0.6.and 0.3. The less polar band on elution with ethylacetate, evaporation and crystallization from acetonehexane gives 20 mg.of 12,8,15a-diacetoxy-A-norprogesterone and the more polar band onsimilar work-up gives 70 mg. of 12,6,15a-dihydroxy-A-norprogesteroneIS-acetate having a melting point about 172174 (another crystalline formmelting at about 220222 C. is also obtained), [M +17.9' (chloroform),

R211; 233 my (6, 16,800), A231? 2.92, 5.88, 6.18 1.27 (S, 3-H) e)4 UDOREXAMPLE 3- 5a-a1cet0xy Z-keto-A -norp rogesterone To a stirred solutionof 100 mg. of 12,6,15a-dihydroxy- A-norprogesterone 15-acetate in ml. ofreagent grade acetone, 0.9- ml. of an aqueous solution containing 20 mg.f'chron1ic anhydride and 32mg. of sulfuric acid per milliiter is addeddropwise. After five minutes a few drops of nethonal are added and themixture diluted with water and. extracted with chloroform. Thechloroform is washed vith water and evaporated in vacuo. Crystallizationof the 'esidue from acetone-hexane gives 86 mg. ofl5a-acetoxyl2-keto-A-norprogesterone having a melting point about.68-170; [11], +105 (chloroform),

Eli... 232 ma 17,000), M1311 5.73, 5.81, 5.86, and 613 93? 4.23 (s,3-H),

1.90. (15,BH), 7.73 (S, 21-CH 7.99 (S, 15oc-OAC), 8.73 s, 19-CH 8.91 s,18-CH Analysis.-Calcd for C H O (372.44): C, 70.94; H, .58. Found: C,70.95; H, 7.72.

4 EXAMPLE 4 IZ-ketO-ISa-hydrOxy-A-n0rpr0gester0ne 7.73 (S, 21-CH3), 8.72(S, 19CH 8.94 (S, 18-CH Analysis.--Calcd for C H O (330.41): C, 72.70;H, 7.93. Found: C, 72.74; H, 7.99.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

What is claimed is:

1. A compound selected from the group consisting of steroids of theformula wherein Y is hydrogen and X is selected from, the groupconsisting of hydroxy and acyloxy; wherein the acyl radi cal is of ahydrocarbon carboxylic acid of less than twelve carbon atoms.

2. 112B,15a-dihydroxy-A-norprogesterone IS-acetate.

3. 15a-acetoxy-12-keto-A-norprogesterone.

4. 1 2-keto-15u-hydroxy-A-norprogesterone.

References Cited UNITED STATES PATENTS 3/1965 Fried 260-488 4/1965Weisenborn et al 260586 OTHER REFERENCES Schubert et al., Leitschriftfur Naturforschung, vol. 17b, No. 7, July 1962, pp. 436-439.

LORRAINE A. WEINBERGER, Primary Examiner. VIVIAN GARNER, AssistantExaminer.

1. A COMPOUND SELECTED FROM THE GROUP CONSISTING OF STEROIDS OF THEFORMULA